Models of chronic and acute inflammatory diseases

ABSTRACT

Methods and compositions are provided for the creation and screening of non-human animal models having chronic inflammation. Immunocompromised host animals are injected with a population of immunocompetent effector cells, depleted of CD25+ T cells. The effector cells are tolerant of the host major histocompatibility antigens, but reactive to at least one antigen present in the host animal. The transferred cells are preferably stimulated and localized by administration of an immunostimulant at a local site. The animals are useful for a variety of screening assays and for investigation into disease causes and pathways. A variety of chronic inflammatory diseases may be studied with this model, including psoriasis, rheumatoid arthritis, diabetes, inflammatory bowel disease and multiple sclerosis.

BACKGROUND OF THE INVENTION

[0001] Despite recent advances in genomic sequencing efforts, as well asin the fields of pre-clinical drug screening/development and clinicaltrial design, the transfer of existing “pre-clinical” knowledge into theclinic is still very difficult. This is mainly due to the sparseknowledge of the events that occur during the initiation, theperpetuation and the maintenance of inflammatory disease states inhumans.

[0002] The reasons for such incomplete and often low quality informationare numerous: humans cannot intentionally be studied in the pre-clinicalphase, cell isolation is difficult from human tissue, the startingevents of an autoimmune reaction occur without notice, and patients withautoimmune or other inflammatory diseases may not wish to be treated asexperimental subjects. As a result, there is a lack of reliableinformation on which to base decisions about clinical trials. Whenclinical symptoms arise and treatment is required, rational selectionfrom among the many potential anti-inflammatory compounds orcombinations thereof is difficult.

[0003] In order to identify new and useful drugs, screening assays mustbe able to provide biologically relevant information, so that there is agood correlation between the information generated by the screeningassay and the pharmaceutical effectiveness of the compound. Some of themore important features for pharmaceutical effectiveness are specificityfor the targeted cell or disease, a lack of toxicity at relevantdosages, and specific activity of the compound against its molecular orcellular target.

[0004] Inflammatory conditions, particularly chronic inflammatorydiseases, are of particular interest. These diseases are caused by theaction of the immune system, including the inappropriate activation of Tcells, expression of regulatory cytokines and chemokines, loss of immunetolerance, and the like. Modulation of the immune response varies withthe specific factors produced, and the receptors present on theresponding cell.

[0005] Among these diseases are autoimmune and/or chronic inflammatorydiseases, which include multiple sclerosis and inflammatory boweldiseases (IBD, ulcerative colitis and Crohn's disease), colitis,diseases of the joints, such as rheumatoid arthritis, attacks on nucleicacids, as observed with systemic lupus erythematosus and such otherdiseases as psoriasis, insulin dependent diabetes mellitus (IDDM),Sjogren's disease, myasthenia gravis, thyroid disease, Alzheimerdisease, uveitis, and cardiovascular diseases.

[0006] The initiating step in autoimmune disease pathology is stillmysterious in many cases, particularly in humans where the diseases arelargely sporadic, and symptoms may appear years after the first T celllaunches its attack. It has therefore been difficult to design effectivetherapies that prevent initiation of disease, although there are commonfeatures in many of the later stages of disease. Inflammation at thesite of the disease is often found, caused by the release ofinflammatory cytokines by T cells and other pro-inflammatory cells (e.g.macrophages, dendritic cells, B cells, NK cells), and accompanied by thedestruction of autologous cells.

[0007] Recent studies using murine models of experimental chronicinflammation are defining the nature of the immunological disturbancesthat initiate inflammation and destruction of specific organs (forexample, see Mombaerts et al. Cell, 1993. 75(2): p. 274-82; Tarrant etal. J Immunol, 1998. 161(1): p. 122-7; Powrie et al. Immunity, 1994. 1:p. 553-562; Hong et al. J Immunol, 1999. 162(12): p. 7480-91; Horak,Clin Immunol Immunopathol, 1995. 76(3 Pt 2): p. S172-3; Ehrhardt et al.J Immunol, 1997. 158(2): p. 566-73; Davidson et al., J Immunol, 1998.161(6): p. 3143-9; Kuhn et al. Cell, 1993. 75(2): p. 263-74; Neurath etal., J Exp Med, 1995 182(5): p. 1281-90). Increased understanding ofdisease promoting inflammatory cells is providing insights into themechanism controlling the immune responses within target organs.

[0008] Evidence has been presented in the literature for the involvementof different T cell subsets in the development of disease. An importantrole for a distinct T cell population including regulatory and/orsuppressor T cells in maintaining the physical integrity of organspecific immunity has been suggested by recent several studies(Suri-Payer et al., J Immunol, 1998. 160(3): p. 1212-8; Shevach et al.,Novartis Found Symp, 1998. 215: p. 200-11). These investigators andothers (Shimizu et al, J Immunol, 1999. 163(10): p. 5211-8; Itoh et al.,J Immunol, 1999. 162(9): p. 5317-26; Sakaguchi et al. J Immunol, 1995.155(3): p. 1151-64; Takahashi et al., Int Immunol, 1998. 10(12): p.1969-80) have postulated that CD4+ CD25+ T cells play a crucial role inthe suppression of immune responses and one might postulate if a cellpopulation is transferred into an immunodeficient mouse without itssuppressor CD25+ subset, autoimmunity can occur at multiple sites of thebody. This presumes of course that autoimmune causing effector cells areable to reach their target organ. Such an effector cell permissiveenvironment is probably created through the upregulation of adhesionmolecules (Berg et al., Immunol Rev, 1989. 108: p. 5-18; von Andrian etal., Proc Natl Acad Sci USA, 1991. 88(17): p. 7538-42; Berg et al., JExp Med, 1991. 174(6): p. 1461-6; Picker et al. J Immunol, 1990.145(10): p. 3247-55) and the secretion of chemokines (Baggiolini,Nature, 1998. 392(6676): p. 565-8) on the affected tissues, and onendothelial cells allowing the entrance and retention of effector cellsinto the tissue.

[0009] To study the regulatory effects of T cells and otherimmunocompetent cells, animal models have provided a very good tool inthe past. An essential role for the study of human autoimmune conditionswas played in particular by the scid/scid CD4⁺CD45Rb^(hi) cell transfermodel. Over the last decade this model has proven to be a viablescientific tool for the study of dysregulated immune responses, andmoreover, has been proven to be a good tool for the discovery andevaluation of treatment/drug targets, candidates for inflammatory boweldisease and recently psoriasis (Hong et al., supra.; Powrie et al., JExp Med, 1996. 183(6): p. 2669-74; Schon et al., Nat Med, 1997. 3(2): p.183-8). Notably, not only do these animal models resemble humanhistology and physiology in some ways or another, but have been helpfulin determining novel treatment strategies in humans for both diseases.

[0010] One major disadvantage of conventional animal models is that theyare very labor-intensive and costly and thus do not permit largethroughput drug screening. Unfortunately, in vitro screening techniquesare limited in their predictive power. Thus, despite today's advances inpre-clinical science, hard decisions must be made without completepre-clinical, in vivo data.

[0011] With drug discovery moving from target identification tovalidations, reliable biological systems are necessary to confirm,validate and support the recent explosion in the number of potential newdrug targets and drug compounds. The development of robust, reproducibleand scaleable animal models that physiologically resemble human diseaseis very desirable; i.e. models in which the inflammation is trulychronic in nature and the histology that of human, and can be used astreatment models and not only preventive ones. Such animal models mustposses the utility to rapidly advance experimental drug leads rapidlyand reliably in a semi- to high through-put fashion, leading to novel,effective and safe therapeutics.

SUMMARY OF THE INVENTION

[0012] Models are provided for chronic inflammatory diseases. The modelsare useful for testing and screening of biologically active agents forthe treatment of chronic and acute inflammatory disease. A cellpopulation comprising immunocompetent effector cells, which lacks CD25⁺suppressor T cells, is transferred into a cellular environment thatlacks CD25⁺ suppressor T cells but contains a T cell antigen.Preferably, an immunostimulant and/or immunomodulatory co-factor and/orT cell antigen is introduced at a targeted site or organ after the Tcell introduction to enhance T cell response and homing. Animals developacute and chronic inflammatory responses at the targeted site, andprovide a useful model for the development of inflammation, and fordrug/gene screening in the prevention and treatment of chronicinflammatory disease in humans.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a graph depicting disease penetration.

[0014]FIGS. 2A and 2B are graphs depicting the disease scalability anddistribution. Each symbol represents a single ear measurement. (n=10).

[0015]FIG. 3 is a graph demonstrating the chronicity of the induceddisease. Error bars=SEM. A group of animals are considered diseased ifaverage ear thickness >25 μm.

[0016]FIG. 4 is a graph illustrating the effects of a co-factor indisease induction. The disease induction protocol was modified toexamine animals with and without LPS co-injection.

[0017]FIG. 5 shows the effects of anti-IL-12 mAb treatment.

[0018]FIG. 6 is a graph depicting antibody screening against theinflammation model of the invention.

[0019]FIG. 7 is a graph depicting the effect of anti-CD43 monoclonalantibody in the inflammation model of the invention.

[0020]FIG. 8 is a graph depicting the effect of an oral compound ondisease progression.

[0021]FIG. 9 shows the effect of cyclosporin on disease progression.

[0022]FIG. 10 shows the effect of different co-factors on diseaseinduction.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0023] Non-human animal models for chronic inflammatory disease areprovided. The animals are particularly suited as models for T cellmediated autoimmune diseases, such as multiple sclerosis, insulindependent diabetes mellitus, rheumatoid arthritis, and the like.

[0024] An immunocompromised host animal is injected with a population ofimmune cells depleted of CD25⁺ cells, from a donor animal of the same orrelated species. The transferred cells may comprise T cells, naturalkiller (NK) cells, monocytes, etc. The cell population may be furtherselected to enrich for T cell types of interest. For example, the callsmay be selected to have the phenotype CD4⁺CD25⁻. The CD25⁺ populationcontains suppressive T cells that act to down-regulate the T cellresponse. By transferring a population of CD25⁻ cells, the immuneresponsiveness of the cells is increased.

[0025] The host and donor animals are matched, and/or tolerant at thehost major histocompatibility antigens, e.g., are of the same MHChaplotype (MHC matched) but the transferred T cells are responsive to atleast one antigen present in the recipient, e.g. are mismatched at oneor more minor antigens, or otherwise responsive to the presence of a Tcell antigen. For example, a mismatch at minor histocompatibility lociprovides the antigenic immune stimulation for development of chronicinflammation. In a preferred embodiment, an immunostimulant/modulatoryco-factor is administered at a targeted site, in order to localize theeffector T cells.

[0026] These animals provide a useful model for the specific pathogenicrequirements of Th1 promoting cytokines and cells. By providing a moreaccurate model for the human disease, potential therapeutics can beevaluated in the animal model for safety and efficacy prior to clinicaltrials. In addition to screening candidate pharmaceutical agents, thesubject animals are useful in determining the role of “triggering”agents in development of disease, the role of specific T cell subsetsand cytokines, and the role of specific antigens in activation andmaintenance of inflammatory T cells.

[0027] Immunocompromised mammalian hosts suitable for implantation andhaving the desired immune incapacity exist or can be created. Thesignificant factor is that the immunocompromised host is incapable ofmounting an immune response against the introduced pathogenic effector Tcells. Preferred host animals will lack CD25+ T cells. Of particularinterest are small mammals, e.g. rabbits, gerbils, hamsters, guineapigs, etc., particularly rodents, e.g. mouse and rat, which areimmunocompromised due to a genetic defect that results in an inabilityto undergo germline DNA rearrangement at the loci encodingimmunoglobulins and T-cell antigen receptors or to a genetic defect inthymus development (nu/nu).

[0028] Presently available hosts include mice that have been geneticallyengineered by transgenic disruption to lack the recombinase functionassociated with RAG-1 and/or RAG-2 (e.g. commercially available TIM™RAG-2 transgenic), to lack Class I and/or Class II MHC antigens (e.g.the commercially available C1D and C2D transgenic strains), or to lackexpression of the Bcl-2 proto-oncogene. Of particular interest are micethat have a homozygous mutation at the scid locus, causing a severecombined immunodeficiency which is manifested by a lack of functionallyrecombined immunoglobulin and T-cell receptor genes. The scid/scidmutation is available or may be bred into a number of different geneticbackgrounds, e.g. CB.17, ICR (outbred), C3H, BALB/c, C57BI/6, AKR, BA,B10, 129, etc. Other mice which are useful as recipients are NODscid/scid; SGB scid/scid, bh/bh; CB.17 scid/hr; NIH-3 bg/nu/xid and METAnu/nu. Transgenic mice, rats and pigs are available which lackfunctional B cells and T cells due to a homozygous disruption in theCD3_(ε) gene. Immunocompromised rats include HsdHan:RNU-rnu;HsdHan:RNU-rnu/+; HsdHan:NZNU-rnu; HsdHan,:NZNU-rnu/+; LEW/HanHsd-rnu;LEW/HanHsd-rnu/+; WAG/HanHsd-rnu and WAG/HanHsd-rnu/+.

[0029] Generally, the host will be at least about four weeks old. Forexample, mice are often used at about 4 to 12 weeks of age. Themammalian host will be grown in conventional ways. Depending on thedegree of immunocompromised status of the mammalian host, it may beprotected to varying degrees from infection. An aseptic environment isindicated. Prophylactic antibiosis may be used for protection frominfection. Alternatively, it may be satisfactory to isolate thepotential hosts from other animals in gnotobiotic environments aftercesarean derivation. The feeding and maintenance of the host will forthe most part follow gnotobiotic techniques.

[0030] The major histocompatibility locus haplotype of the host animalis determined either through conventional typing methods, e.g. whereoutbred animals are used, or from known information concerning thegenetic characteristics of the animal. In mice, the genes of the majorhistocompatibility locus (MHC) have been very well characterized. TheMHC region is comprised of a number of genes, of which at least fivecontribute to acute graft rejection and graft vs. host disease. Thespecific MHC genes of interest include the class I antigens: H2-K, H2-D,and H2-L; and the class II antigens: H2 I region, which includes H2-Aa,Ab, BI, Ea, Eb, Eb2, Ob, and Pb. Specific information on the haplotypeof most known mouse strains may be found in Klein et al. (1983)Immunogenetics 17(6):553-96.

[0031] The immunocompromised host animals are injected with a cellpopulation comprising immunocompetent T cells, and lacking CD25⁺ cells.Conveniently, a cell population is depleted by reagents specific forCD25 (negative selection), e.g. anti-CD25 antibodies, by flow cytometry,magnetic bead depletion, etc. Alternatively, T cell populationsnaturally deficient in CD25 expression, or deficient through genetargeting from CD25 knockout mice may be used.

[0032] The T cells may be from an allogeneic or xenogeneic donor, andare tolerant to the major histocompatibility antigens of the recipient,but immunoreactive with an antigen present in the recipient, e.g. a Tcall antigen provided by viral infection of the recipient, chronicinfection with a bacterial or protozoan pathogen, sustained release ofan antigenic compound, the presence of one or more minorhistocompatibility antigens of the recipient, etc. By tolerant is meantthat when mixed with appropriate cells (e.g., irradiated lymphocytes)from the recipient, the donor T cells proliferate to a substantiallylesser extent (e.g., <about 10% to 25%) than in an analogous mixedlymphocyte reaction between MHC mismatched cells.

[0033] In contrast to the MHC locus, there are many minorhistocompatibility antigen loci dispersed throughout the genome. Minorantigens generally result from the presentation of cellular proteins onthe surface of cells in conjunction with self MHC. Therefore, virtuallyany protein that is expressed by the host, processed and presented inthe context of MHC antigens, and is polymorphic between host and donor,can serve as a minor histocompatibility antigen. Where there is apersistent or chronic infection, epitopes relating to the infectiousagent can serve as minor histocompatibility antigens. It has beensuggested that some cutaneous antigens may serve as a trigger forchronic inflammatory disease (e.g. H-40, described by Forman et al.(1984) J. Exp. Med. 159:1724-1740; and other antigens described by Changet al. (1994) P.N.A.S. 91:9282-9286; or Menssen et al. (1995) J.Immunol. 155:4078-4083). The subject animals are valuable models fordetermining the role of specific genetic loci in contributing to thedevelopment of inflammatory disease. Such screening may utilize animalsthat are mismatched only at the loci of interest, and then determiningwhether the difference is sufficient for induction of disease.

[0034] There are a number of suitable animals to use as the source of Tcells. In most cases the donor and recipient will be of the samespecies, although for some purposes xenogeneic donors may be used. Inone embodiment of the invention, the donor is allogeneic but is matchedat the MHC locus. For example, congenic mouse and rat strains areavailable that are isogenic at the MHC locus, but have a differentgenetic background. Alternatively, a parental strain may be used as adonor, while an F1 animal acts as recipient, e.g. a BALB/c donor into aBALB/c×C57bl/6 recipient. Alternatively, syngenic cells can be used inthe presence of other exogenous T cell antigen(s) in the hostenvironment, e.g. proteins, peptides, endotoxins, superantigens, and thelike. Alternatively, CB57/BL6 mice can be used as donors, and the donorcells can be transferred into RAG-1™ and/or RAG-2™ deficient mice.

[0035] Alternatively, one may use a chimeric animal as the source ofdonor cells. For example, one can create a chimera by transferringhematopoietic stem cells (HSC) into a recipient, where the HSC are of adifferent genotype than the recipient. The HSC then differentiate into Tcells which are “educated” in the thymus, and so are restricted to therecipient MHC type. These cells from the chimera can then be harvestedand used in the subject methods, because they are both tolerant andrestricted to the MHC type of the thymus. It will be understood by oneof skill in the art that the thymic MHC in this example must becompatible with the ultimate recipient animal. This procedure can alsobe used to create xenogeneic chimeras (see for example, U.S. Pat. No.5,625,127), allowing the use of human cells in the subject methods.

[0036] The injected cell population comprises immunocompetent T cells,and may also comprise other CD25 negative hematopoietic cells, includingmacrophages, B cells, monocytes, etc. T cells are conveniently isolatedfrom secondary immune organs, e.g. spleen, lymph node, thymus, etc. Forexample, an unfractionated suspension of spleen cells, lymph node, etc.can be depleted of CD25⁺ cells and injected into the animal. Cells mayalso be isolated from peripheral blood, cord blood, apheresis product,etc. Cell populations may be enriched for various cell fractions ofinterest, e.g. by density gradient, elutriation, cell sorting, etc. Inone embodiment of the invention, the population is selected for CD4positive cells, which enriches for T helper cells. In another embodimentof the invention, the cell population is depleted of hematopoietic andlymphoid progenitor cells, as known in the art, in order to decrease thepossibility of de novo T cell maturation in the host animal.

[0037] In another embodiment, the CD25 depleted cell population ispre-incubated with antigen presenting cells, which may be syngeneic,allogeneic, xenogeneic, usually comprising an exogenous antigen to whichthe CD25 depleted population is responsive; having mismatches at minorMHC loci; and the like. Optionally, pro-inflammatory factors, e.g.lymphokines, endotoxins, superantigens etc.; or antibodies againstsuppressor factors, e.g. TGF-β or IL-10, etc. are present. The cells areincubated for a period of time sufficient to induce an immune response,and are then introduced into a normal non-immunocompromised orimmunocompromised host. In another embodiment, whole cells are incubatedwith pro-inflammatory cytokines that down-regulate CD25 expression on Tcells and then are introduced into the host environment, e.g.non-immunocompromised or immunocompromised, syngenic or minor-haplotypemismatched.

[0038] Inflammatory diseases Can also be transferred from one animalexpressing disease to another naive animal by extracting effector cellsfrom the diseased animal and injecting them into multiple naive animals.In another embodiment, a secondary transfer is performed, where wholespleen or lymph node cells from a primary host that was previouslytreated with a CD25 depleted population, as described above, aretransferred into a secondary host. The primary host may be diseased ornot-diseased. The cells from the primary host may be unfractionatedspleen, lymph node, etc., or may be depleted of CD25 positive cells.Effector cells can be found in secondary lymphoid tissue, especiallyspleen but also draining lymph node, and the actual diseased organtissue.

[0039] Separation of the desired cells for engraftment will generallyuse affinity separation to provide a substantially CD25 negativepopulation, usually comprising after separation not more than about 5%CD25⁺ cells, more usually not more than about 3% CD25⁺ cells, and may beless than about 1% CD25⁺ cells. Techniques for affinity separation mayinclude magnetic separation, using antibody-coated magnetic beads,affinity chromatography, cytotoxic agents joined to a monoclonalantibody or used in conjunction with a monoclonal antibody, e.g.complement and cytotoxins, and “panning” with antibody attached to asolid matrix, e.g. plate, or other convenient technique. Techniquesproviding accurate separation include fluorescence activated cellsorters, which can have varying degrees of sophistication, such asmultiple color channels, low angle and obtuse light scattering detectingchannels, impedance channels, etc. The cells may be selected againstdead cells by employing dyes associated with dead cells (propidiumiodide, LDS). Any technique may be employed which is not undulydetrimental to the viability of the selected cells.

[0040] The affinity reagents may be specific receptors or ligands forthe cell surface molecules indicated above. In addition to antibodyreagents, peptide-MHC antigen and T cell receptor pairs may be used;peptide ligands and receptor; ligand and receptor molecules, and thelike. Antibodies and T cell receptors may be monoclonal or polyclonal,and may be produced by transgenic animals, immunized animals,immortalized human or animal B-cells, cells transfected with DNA vectorsencoding the antibody or T cell receptor, etc. The details of thepreparation of antibodies and their suitability for use as specificbinding agents are well-known to those skilled in the art.

[0041] Of particular interest is the use of antibodies as affinityreagents. Conveniently, these antibodies are conjugated with a label foruse in separation or used in conjunction with a labeled second antibodythat binds to them. Labels include magnetic beads, which allow fordirect separation; biotin, which can be bound to avidin or streptavidinbound to a support; fluorochromes, which can be used with a fluorescenceactivated cell sorter; or the like, to allow for ease of separation ofthe particular cell type. Fluorochromes that find use includephycobiliproteins, e.g. phycoerythrin and allophycocyanins, fluoresceinand Texas red.

[0042] The antibodies are added to a suspension of lymphocytes, andincubated for a period of time sufficient to bind the available cellsurface antigens. The incubation will usually be at least about 5minutes and usually less than about 30 minutes. It is desirable to havea sufficient concentration of antibodies in the reaction mixture so thatthe efficiency of the separation is not limited by lack of antibody. Theappropriate concentration is determined by titration. The medium inwhich the cells are separated will be any medium which maintains theviability of the cells and binding of antibody. A preferred medium isphosphate buffered saline containing from 0.1 to 0.5% BSA. Various mediaare commercially available and may be used according to the nature ofthe cells, including Dulbecco's Modified Eagle Medium (DMEM), Hank'sBasic Salt Solution (HBSS), Dulbecco's phosphate buffered saline (DPBS),RPMI, Iscove's medium, PBS with 5 mM EDTA, etc., frequently supplementedwith fetal calf serum, BSA, HSA, etc.

[0043] The labeled cells are then separated as to the expression ofCD25, and optionally CD4. The separated cells may be collected in anyappropriate medium that maintains the viability of the cells, usuallyhaving a cushion of serum at the bottom of the collection tube. Variousmedia are commercially available and may be used according to the natureof the cells, including DMEM, HBSS, DPBS, RPMI, Iscove's medium, etc.,frequently supplemented with fetal calf serum.

[0044] Compositions enriched for the desired T cells are achieved inthis manner. 90% of CD25pos T cells are depleted in the finalCD25negative T cell population. The enriched cell population may be usedimmediately, or may be frozen at liquid nitrogen temperatures and storedfor long periods of time, being thawed for use when needed. The frozencells will usually be stored in 10% DMSO, 10-90% FCS, 40% RPMI 1640 orother medium. Once thawed, the calls may optionally be expanded by useof growth factors or stromal cells associated with T cell proliferationand differentiation.

[0045] The population of purified T cells are injected into theimmunocompromised recipient. Routes of administration include systemicinjection, e.g. intravascular, subcutaneous, or intraperitonealinjection. Where the recipient animal is a mouse, the number of cellsinjected will usually be at least about 0.5×10⁵ and not more than about5×10⁵, more usually at least about 1×10⁵, preferably between about 3×10⁵and 4×10⁵. Where the recipient animal is a larger animal, the number ofcells will be increased accordingly.

[0046] Preferably, after transfer of the T cell population, a localizedimmunostimulant and/or immunomodulating co-factor is delivered in orderto facilitate localization, retention and replication of the effector,disease causing T cells. An immunostimulant or immunomodulator can beany agent that can contribute or induce either directly or indirectlyinflammation through the release of cytokines, lymphokines and theupregulation of adhesion molecules. To accomplish this, the co-factor isadministered, generally at a localized site, following transfer of the Tcells. The timing of administration is varied depending on the desiredeffect, but is generally performed from 1 day to 1 week after T celltransfer. Many immunostimulants are known in the art, including LPS andendotoxins in small doses, alpha interferons, interleukin-1, modifiedtumor necrosis factor, CD40 ligand, poly IC, virus, etc.

[0047] In one embodiment of the invention, the immunostimulatoryco-factor is a virus or viral vector, e.g. adenovirus, vaccinia, HSV,SV40, and AAV, etc. The immuno-stimulatory effect may be provided by theviral coat proteins present on the virus particles, and/or by viralproteins or other genes expressed upon infection of the target cell.Live virus is not required for the co-factor effect, killed virus orvector encoding viral proteins are also suitable. Suitable systems aredisclosed, for example, in Fisher-Hoch et al., PNAS 86:317-321, 1989;Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al.,Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988;Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., PNAS91:215-219, 1994; Kass-Eisler et al., PNAS 90:11498-11502, 1993; Guzmanet al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res.73:1202-1207, 1993. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.The DNA may also be “naked,” as described, for example, in published PCTapplication WO 90/11092, and Ulmer et al., Science 259:1745-1749, 1993.The uptake of naked DNA may be increased by coating the DNA ontobiodegradable beads, which are efficiently transported into the cells.In addition to viral genes, vectors and viruses can be modified toencode immunomodulatory genes, e.g. IL-2, IL-12, CD40, IFN-gamma,GM-CSF, TNF-alpha, etc.

[0048] The immunostimulant is administered to the host in the mannerconventional for the particular composition, generally as a single unitdose in buffered saline, optionally combined with an adjuvantformulation, where booster doses, typically one to several weeks later,may additionally be delivered enterally or parenterally, e.g.,subcutaneously, cutaneously, intramuscularly, intradermally,intravenously, intraarterially, intraperitoneally, intranasally, orally,intraheart, intrapancreas, intraarticular, etc. Localization can beachieved by administration at the targeted site, use of sustainedrelease implants, delivery in the form of non-diffusible particles, andthe like, as known in the art.

[0049] In one embodiment of the invention, the immunostimulant is apolyclonal activating agent, which may include endotoxins, e.g.lipopolysaccharide (LPS); and superantigens (exotoxins) (see Herman etal. (1991) Annu Rev Immunol 9:745-72). Endotoxin primarily interactswith CD14 receptors on macrophages, while superantigens preferentiallyactivate T cells. Both cell types are thus triggered to releasepro-inflammatory cytokines. Superantigens (SAgs) are presented by majorhistocompatibility complex (MHC) class II molecules and interact with alarge number of T cells expressing specific T cell receptor V betadomains. SAgs may be endogenous, e.g. MIs; bacterial, e.g. SEB, SEA; orviral, e.g. mouse mammary tumour virus.

[0050] Alternatively, one may use immunostimulatory polynucleotidesequences (ISS). The use of these sequences is known in the art, forexamples see Bauer et al. (1999) Immunology 97(4):699-705; Klinman etal. (1999) Vaccine 17(1):19-25; Hasan et al. (1999) J Immunol Methods229(1-2):1-22; and others. For example, an “immunostimulatoryoligonucleotide” has been described as an oligonucleotide that containsa cytosine/guanine dinucleotide sequence and stimulates maturation andactivation of DC. An immunostimulatory oligonucleotide of interest maybe between 2 to 100 base pairs in size and typically contain a consensusmitogenic CpG motif represented by the formula: 5′ X₁ X₂ CGX₃ X₄ 3′,where C and G are unmethylated, X₁, X₂, X₃ and X₄ are nucleotides and aGCG trinucleotide sequence is not present at or near the 5′ and 3′termini (see U.S. Pat. No. 6,008,200, Krieg et al., issued Dec. 28,1999, herein incorporated by reference).

[0051] Preferably the immunostimulatory oligonucleotides range between 8to 40 base pairs in size. In addition, the immunostimulatoryoligonucleotides are preferably stabilized oligonucleotides,particularly preferred are phosphorothioate stabilized oligonucleotides.In one embodiment, X₁ X₂ is the dinucleotide GpA. In another embodimentX₃ X₄ is the dinucleotide TpC or TpT.

[0052] The dose and protocol for delivery of the immunostimulant willvary With the specific agent that is selected. Typically one or moredoses are administered. One particular advantage of the use of ISS inthe methods of the invention is that ISS exert immunomodulatory activityeven at relatively low dosages. Although the dosage used will varydepending on the clinical goals to be achieved, a suitable dosage rangeis one which provides from about 1 Fg to about 10,000 Fg, usually atleast about 1,000 Fg of ISS in a single dosage. Alternatively, a targetdosage of ISS can be considered to be about 1-10 femtomole in a sampleof host blood drawn within the first 24-48 hours after administration ofISS. Based on current studies, ISS are believed to have little or notoxicity at these dosage levels.

[0053] In an alternative embodiment, a non-replicating virus or viralcoat protein is used as the immunostimulant. Virions of interest includeherpes viruses, e.g. HSV, EBV, CMV, etc.; adenoviruses, e.g. E1 deletedadenovirus; retroviruses; etc. The virus may optionally comprise amarker gene, such as lacZ, in order to track efficiency of infection.For examples, see Byrnes et al. (1995) Neuroscience 66(4):1015-24; Woodet al. (1994) Gene Ther 1(5):283-91; and Kajiwara et al. (1997) Hum GeneTher 8(3):253-65.

[0054] Injection of a non-replicating virus leads to an inflammatoryresponse, e.g. in brain or neural tissue. Much of this inflammation isinduced directly by the virion particles themselves rather than throughthe expression of new proteins from the virus. By two days there is alarge increase in major histocompatibility complex class I andP-selectin expression and a heavy infiltration of leukocytes, mainlymacrophages and T cells.

[0055] In an alternative embodiment, the CD25 depleted cells areintroduced into a pro-inflammatory environment either before or duringin vivo introduction to the host. A pro-inflammatory environment can beinduced by adding pro-inflammatory factors or antibodies againstanti-inflammatory (suppressor) factors, e.g. IFN-γ, IL-12, TNF-alpha,anti-TGF-beta, anti-IL-10, in vivo and/or in vitro prior to introductioninto an animal.

[0056] After administration of the T cells and co-factor, within about 4to 8 weeks the animals develop chronic inflammatory disease. Scoring ofthe disease severity is based on physical appearance, measurable earthickness, cytokine expression, presence of T cells at the lesion, etc.A more detailed analysis may utilize histological section of varioustissues, conveniently ear, eyelid, tail, etc. Specific histologicalfeatures include mononuclear cell infiltration; high vascular density;etc.

[0057] To more fully characterize the disease, immunophenotypic analysismay be performed to detect a variety of relevant antigenic determinants.To characterize the types of immune cells present, immunohistochemicalstains for various leukocyte markers may be performed. The expression ofadditional adhesion molecules that are relevant to the pathophysiologyof chronic inflammatory disease may include mononuclear cell infiltrate;T cells at lesions; and the expression in adjacent blood vessels offocal E-selectin, P-selectin, ICAM-1 and diffuse vascular cell adhesionmolecule-1 (VCAM-1) expression.

[0058] The subject animals are useful for screening candidatetherapeutic agents and treatment modalities. Through use of the subjectanimals or cells derived therefrom, one can identify ligands orsubstrates that affect the progression of chronic inflammatory disease.Of particular interest are screening assays for agents that have a lowtoxicity for human cells.

[0059] Drug screening protocols will generally include a panel ofanimals, for example a test compound or combination of test compounds,and negative and/or positive controls, where the positive controls maybe known immunosuppressive agents. Such panels may be treated inparallel, or the results of a screening assay may be compared to areference database.

[0060] A wide variety of assays may be used for this purpose, includinghistological analysis of effectiveness, determination of thelocalization of drugs after administration, labeled in vitroprotein-protein binding assays, protein-DNA binding assays,electrophoretic mobility shift assays, immunoassays for protein binding,and the like. Depending on the particular assay, whole animals may beused, or cells derived therefrom, particularly skin cells, e.g.keratinocytes. Cells may be freshly isolated from an animal, or may beimmortalized in culture. Candidate therapies may be novel, ormodifications of existing treatment options.

[0061] For screening assays that use whole animals, a candidate agent ortreatment is applied to the subject animals. Typically, a group ofanimals is used as a negative, untreated or placebo-treated control, anda test group is treated with the candidate therapy. Generally aplurality of assays are run in parallel with different agent dose levelsto obtain a differential response to the various dosages. The dosagesand routes of administration are determined by the specific compound ortreatment to be tested, and will depend on the specific formulation,stability of the candidate agent, response of the animal, etc.

[0062] The analysis may be directed towards determining effectiveness inprevention of disease induction, where the treatment is administeredbefore induction of the disease, i.e. prior to injection of the T cellsand/or pro-inflammatory cytokine. Alternatively, the analysis isdirected toward regression of existing lesions, and the treatment isadministered after initial onset of the disease, or establishment ofmoderate to severe disease. Frequently, treatment effective forprevention is also effective in regressing the disease.

[0063] In either case, after a period of time sufficient for thedevelopment or regression of the disease, the animals are assessed forimpact of the treatment, by visual, histological, immunohistological,and other assays suitable for determining effectiveness of thetreatment. The results may be expressed on a semi-quantitative orquantitative scale in order to provide a basis for statistical analysisof the results.

[0064] The term “agent” as used herein describes any molecule, e.g.protein or pharmaceutical, with the capability of affecting the severityof chronic inflammatory disease. An agent or treatment, e.g. UV light,is administered to an animal of the invention, or to cells derivedtherefrom. Antibodies specific for cytokines, polyclonal activatingagents, and T cell antigens are agents of particular interest. Mostpreferably, according to another aspect of the instant invention, theagents are monoclonal antibodies, e.g. which neutralize lymphokines orblock adhesion molecules.

[0065] Other candidate agents encompass numerous chemical classes,typically organic molecules. Candidate agents comprise functional groupsnecessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The candidate agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Candidateagents are also found among biomolecules including, but not limited to:peptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof.

[0066] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

[0067] The therapeutic agents may be administered to patients in avariety of ways, orally, topically, parenterally e.g. subcutaneously,intramuscularly, intravascularly, etc. Depending upon the manner ofintroduction, the compounds may be formulated in a variety of ways. Theconcentration of therapeutically active agent in the formulatedpharmaceutical compositions may vary from about 0.1-100 wt. %.

[0068] The pharmaceutical compositions can be prepared in various forms,such as granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

[0069] It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,constructs, and reagents described, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which scope will be determined by thelanguage in the claims.

[0070] It must be noted that as used herein and in the appended claims,the singular forms “a”, “and”, and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a mouse” includes a plurality of such mice and reference to “thecytokine” includes reference to one or more cytokines and equivalentsthereof known to those skilled in the art, and so forth.

[0071] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this invention belongs. Although any methods,devices and materials similar or equivalent to those described hereincan be used in the practice or testing of the invention, the preferredmethods, devices and materials are now described.

[0072] All publications mentioned herein are incorporated herein byreference for all relevant purposes, e.g., the purpose of describing anddisclosing, for example, the cell lines, constructs, and methodologiesthat are described in the publications which might be used in connectionwith the presently described invention. The publications discussed aboveand throughout the text are provided solely for their disclosure priorto the filing date of the present application. Nothing herein is to beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

[0073] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the subject invention, and are not intended to limitthe scope of what is regarded as the invention. Efforts have been madeto ensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

EXPERIMENTAL

[0074] Animal Model for Chronic Inflammation

[0075] Mice. Female Balb/c mice (donor mice) were purchased from JacksonLabs (Bar Harbor, Me.) or similar source, and C.B-17/lcr scid/scid(recipient mice) were purchased from Taconic (Germantown, N.Y.) orCharles River. All mice were housed in a specific pathogen freeenvironment and were used between 4-8 wk of age. Mice were housed 2-5per microisolator. All scid/scid mice were handled with gloves under aclass II hood, fed sterile food and water ad libitum, and maintained insterilized microisolators that are changed twice weekly. Donor mice werehoused in conventional cages that were changed weekly.

[0076] Induction of chronic skin inflammation. Briefly, splenocytes werecollected from 5-10 week old donor mice (Balb/c) and donor populationwas either enriched for CD4+ and depleted of CD25+ cells, or depleted ofCD25+ cells only. The collected cell population was injectedsubcutaneously (s.c.) into C.B-17/lcr scid/scid mice, aged 4-8 weeks(usually 3×10⁵ to 5×10⁵ cells per mouse in 200-400 μL). A systemic(s.c., i.p. or i.v.) injection of an immuno-modulating agent was given24 hours following cell transfer. Alternatively, these co-injectionswere given at the same time to one week after. The co-injection was,alternatively, repeated every other day or once a week for the entirecourse of the experiment.

[0077] Day 0:

[0078] Cell Selection: Balb/c mouse spleens were collected andhomogenized by pressing through 100μ cell strainer (Falcon) andsuspended in cold PBS supplemented with 10% FBS. Cell suspension wasthen centrifuged (400G) and the cell pellet was retained, resuspended in2 ml warm RBC lysing buffer (37 C.) per spleen, and incubated 3 minutesat 37° C. The cells were then washed with cold PBS+10% FBS and againcentrifuged. The cell pellet was then resuspended in 5 ml cold PBS+10%FBS and we added CD4+ selection beads (Dynal) 25 μl per spleen to thecell suspension. This mixture was then incubated for 30 minutes at 4° C.on a rotator and using a magnetic particle collector (MPC) the bead-cellcomplexes were collected and rinsed three times with PBS+10% FCS. Thecell-bead complexes were then resuspended in 5 ml warm media (DME,RPMI)+10% FBS, and 20 μl/spleen CD4 DetachaBead (Dynal) was added andincubated for 45 minutes RT on rotator to remove beads from the cellsurface. We removed beads using a MPC, rinsed them twice with PBS+10%FBS and retained supernatants. The resultant cell quantities were 5-8million cells per spleen. We then resuspend these cells in 1 ml PBS+10%FBS with 8 μl/spleen anti-CD25-Biotin conjugated mAb and incubate 20minutes 4° C. Again, cells were washed and resuspended in 1 mL PBS+10%FBS with 25-30 μl steptavidin beads and incubate for 20 minutes at 4° C.Bead-CD25+ cell complexes were removed using an MPC. The cells were onceagain, collected, washed, and the pellet was checked for stray beadswhich were removed if necessary using MPC.

[0079] Scid/scid mice were then injected with 1×10⁵-5×10⁶ cells in200-400 μl PBS sc. (all SC injections were done with the mice undergeneral anesthesia).

[0080] After 24 hours all recipient mice were given a co-injection of 20μg LPS (or 10 μg SEB) s.c. Disease causing T cells then proliferated andbegan to induce inflammation over a period of 4 weeks.

[0081] Week 4-6: Disease expression period. Beginning on week 4,measurements of skin thickness were taken from both ears to monitorlevel of disease expression and incidence.

[0082] The thickness of the skin on the ear was measured using a Dyermicrometer. The micrometer was first modified to better perform in themeasurement of soft tissue. The contact pads are reduced to 4 mm, andthe spring tension is reduced to <3 lbs.

[0083] Psoriatic mice were selected for use in compound screening basedon ear thickness and clinical phenotype. Mice were then randomlyassigned to experimental groups.

[0084] Week 6-10: Treatment Period (2-4 Weeks)

[0085] Administration of experimental compounds (which includedantibodies, small molecules, chemicals, viral vectors, drugs) wasconducted regularly (once, twice or 3 times per week or daily) for 2-4weeks at a dose relative to appropriate mg/kg dosages. In general,administration of all compounds were given systemically (SC, IP or IV).Along with the experimental compounds, control groups were runsimultaneously with injections of PBS, a negative control (isotypecontrol), and a positive control (anti-IL-12, anti-TNFα,corticosteroids, or other known compounds that result in the resolutionof psoriatic lesions).

[0086] Mice were observed and data recorded for ear thickness and totalbody weight on a weekly basis. Body weight was monitored to help monitorthe overall health of the animal, e.g. exclude viral infection andcolitis.

[0087] Week 8-12: Evaluation Period (1-2 Weeks after Last Injection ofExperimental Compound)

[0088] After the completion of treatment period with the experimentalcompounds, including positive and negative controls a period of at leastone or two weeks was allowed to pass to confirm that the drug did or didnot have an effect on the severity of disease. Thus the time from thefirst injection to the end of this waiting period was generally 3-4weeks in all experiments. At the end of this period, mice weresacrificed, biopsies from both ears taken, and 6 cross sections weremade, stained (H and E) and evaluated in blind fashion by at least 2investigators (given histology score ranged 0-4). Biopsies from allother skin areas were occasionally taken as well.

[0089] Skin biopsies were taken from the ear by removing the earentirely by making the cut below the base of the ear. This method wasrequired to make an adequate observation of the organ as possible Fromthe base of the ear to the tip the tissue tends to become thinner. Oftenmild disease is easier to detect at the base of the ear. The histologyscore was determined by evaluating 6 sections (2 cross sections madefrom the tip, middle and base sections of the ear). Other biopsies areuseful to support the data collected from the ear. Such cases includedextremely severe clinical cases where hair loss occurs indicatinginvolvement of other regions of skin.

[0090] Scid/scid mice engrafted with T cells have been shown in previousstudies to come down with some incidence of colitis. It was found in ourexperiments that this procedure could be used with immunomodulatoryco-factors to create organ specific inflammation, e.g. psoriasis,colitis, etc. Because of the immunostimulating properties of bacterialmitogens or bacterial superantigens it was initially tested whether theco-administration of such agents would have a positive effect on theinduction of disease with this novel scid/scid transfer model.

[0091] Disease induction, severity, and chronicity. Initially theanimals were tested to determine the percentage of mice that came downwith disease. The experimental data represented in FIGS. 1 and 2 comesfrom a group of 40 scid/scid mice that received a transfer of CD4+/CD25−T cells as described above. In, brief, naive scid/scid mice wereinjected sc. with 3-6×10⁵ CD4⁺CD25⁻ cells on day 0, followed by a sc.injection of 20 μg LPS 24 hours later. The mice were then handled withnormal husbandry for 4 at which time clinical signs of psoriasis beginappear on the ears in the form of reddened, thickened skin. On week five50% of the animals were considered diseased (>=25 μm skin thickness).The incidence of disease improved by week 6 to 76%, and reached 96% onweek 7. This data is from one experiment and is representative of 6experiments.

[0092] The psoriasis in this model is significant it does reach highseverity. The normal scid/scid mouse skin thickness in the ear is 18-22μm. As seen in FIG. 1 the distribution of severity attainable in thismodel has a majority of the mice expressing severe levels of disease(Each mark represents a single ear measurement. n=80. Normal earthickness: 19-22, Mild disease 25-30 μm. Moderate disease 31-39 μmSevere disease >40 μm). As seen in FIG. 2, the progress of disease canbe monitored and the severity of disease is scaleable. The mild tomoderately diseased animals are shown to come down with disease (earthickness becomes >25 μm) between week 7 and 8. In mice that have moresevere disease by week 8 will show a more aggressive development ofpsoriasis and will become disease (develop skin thickness >25 μm)starting on week 5.

[0093] In order to determine that this disease was a chronicinflammation, several mice from various experiments were observed forgreater than 14 weeks after cell transfer. In FIG. 3 one group of 10animals which were induced with psoriasis by the standard protocol wereobserved for 17 weeks after the transfer of T cells. Measurements of theears of 10 mice were averaged (20 ears in total). In this experiment theaverage measurement (n=20) was >25 μm on week 5. It was found that thediseased condition not only developed from moderate to severe levels(>30 μm) but also lasted for 12 weeks. This is an adequate time periodto demonstrate that the disease does not resolve itself. The chronicityof disease may be attributed to the autoantigens that drive the disease.It is also observed that the severity of the disease is narrow ranged(between 32-36 μm) for a period of 6 weeks (week 9-15). This data wasfound to be representative of 4 separate experiments.

[0094] Co-factor injections are important for their mitogenicproperties. In one experiment five mice were compared with the standardinduction protocol which included an injection of 20 μg LPS on day oneto 5 mice that received the same cells but were not given the injectionof LPS. Starting on week 6 the mice that received the LPS displayed ahigher severity of psoriasis like disease. By week 7 the mice thatreceived the LPS had an average ear thickness of 36.4 μm±0.7 compared to30 μm±1.2 in the mice that did not receive LPS. Both groups of mice hadan incidence of disease of 100% (5/5), shown in FIG. 4. Data representsthe average of 10 measurements of skin thickness (1 per ear, 2 per mousen=5 mice), Error bars=SEM. p=3.6×10⁻³ Therefore, the data shows that theuse of bacterial antigens results in an increased severity of disease.

[0095] The effect of different co-factors on disease induction wasassessed. All mice were induced with the same cell population describedin the standard protocol. Each group received a single injection ofco-factor after cell transfer as follows. No Co-injection: 200 μl PBSs.c on day 1. LPS: 20 μg LPS s.c. diluted in PBS on day 1. LPS+WholeCells: LPS and 10⁶ CD25⁻ cells s.c. on day 14. Viral Vector: 1×10⁶ viralparticles injected s.c. in 200 μl PBS. SEB: 10 μg SEB diluted in 200 μlPBS injected s.c.

[0096] The viral vector is the Adenovirus serotype 5 with deletion of E1and E3 genes. The transgene is the LacZ gene under the control of thecytomegalovirus-IE promoter. The dose (10⁶ VP) is the virus particlecount, not the infections dose (our virus particle dose is equivalent to10⁵ TCID 50). The CD25⁻ cells were selected from Balb/C spleen withoutother enrichment, and so included all cells including CD8, B, NK cell.The results are shown in FIG. 10. It can be seen that the viral vectorco-factor produced a very strong response.

[0097] Phenotype of disease inducing cells. The sorted CD4⁺/CD25⁻ cellpopulation was tested for purity, by staining with florescein conjugatedantibodies against CD4 and CD25 and analysis using a FacsCalibur (BectonDickenson) and Cell Quest Software. The population was found to stainpositively for CD4 and negatively for CD25 on greater than 97% of thecells.

[0098] The sorted CD4⁺/CD25⁻ cell population was also tested by stainingwith fluorescein conjugated antibodies against CD45RB. It was found thatthe disease inducing cells have a heterogeneous phenotype of CD45RB hiand low. Thus, the induction of disease is CD45 independent.

[0099] Experiments were also performed demonstrating that secondarytransfer of whole spleen cell suspensions from a primary host withdisease induced as described above, results in the transfer of disease.Whole spleens from diseased mice (induced by standard protocol) weretreated with red blood cell lysing buffer, and reinjected into naivescid/scid mice. Each mouse received 2.5×10⁵ cells s.c. (n=3).

[0100] It was further shown that a suspension of unfractionated spleencells depleted of CD25 positive cells can be used to induce disease Thehost animals were injected with 500,000 splenocytes from normal Balb/Cmice, that were depleted of CD25 cells by the same magneticbead-antibody method described above, in combination with LPS. Cellsincluded in this population, in addition to CD4 T cells, are CD8 Tcells, B cells, NK cells, macrophages, dendritic cells etc. The resultwas an induction of disease.

[0101] The general health of the animals with inflammatory disease wasmonitored not only by daily observations but also by measuring theirbody weight. In previous studies, the induction of inflammation ofteninvolved colitis, which results in a general decrease in health of theanimal. We observed three groups of 5 psoriatic mice in each group andfound that even after the induction of the inflammatory disease theaverage weight of the mice stayed very consistent in all groups. Inanother study 5 psoriatic animals with an average ear thickness startingat 35 μm and increasing to 45 μm (moderate to severe severity) werefound to have an average body mass holding between 20-22 mg (normalhealthy body mass 19-23 mg) for the entire course of diseaseprogression, and up to 11 weeks post cell transfer. This demonstratesthat even severely affected psoriatic mice remain healthy for anextended period of time.

[0102] To demonstrate that the disease was not an artifact of themechanical manipulation of the mice, a study was conducted to show thatthe transferred cells were the cause of disease. Naive scid/scid micewere given sc. Injections containing 2.5×10⁵ whole (un-enriched) spleencells from psoriatic mice that had been induced to develop psoriasiswith CD4⁺/CD25⁻ cells. All of the test subjects came down with disease(3 out of 3 at week 8). It may be noted that the spleens from diseasedscid/scid mice are small. This study shows that the cells transferred inthis invention are the cause of disease and that the cells maintaintheir disease causing properties even after multiple animal transfers.

[0103] Administration of anti IL-12 mAb. There have been many examplesof the effective anti-inflammatory effect of anti IL-12 mAb treatment.In animal model of the present invention, it was also found to cause areduction in psoriasis lesions. Animals were tested for the effect oftreatment during ongoing psoriatic disease. The disease was induced bythe standard protocol, and treatment began on week 9. The treatment withthe anti-IL-12 mAb was 1 mg/mouse/week. In the initial experiments (n=5untreated, n=5 treated) mice received injections of anti-IL-12 mAb atweeks 9 and 10, and were observed for 2 weeks after the final injection.Control animals were left untreated, isotype treated animals received anisotype matched monoclonal antibody against a non mouse antigen.Anti-IL-12 treatment resulted in a skin thickness improvement of −7 μmcompared to an increase in skin thickness of +2 μm in control isotypetreated animals and untreated animals, as shown in FIG. 7.

[0104] In a separate study, anti IL-12 mAb was used in a positivecontrol group, to determine if an experimental anti-mouse antigenantibody had anti-inflammatory properties. In this experiment there were5 mice per experimental group: positive control (anti IL-12), negativecontrol (isotype matched anti Human antigen mAb), and the experimentalmAb labeled BSK Ab001. At week 12 the animals were sacrificed forhistology of the skin tissue. Where the isotype control mice had anaverage histology score of 2.7 on a scale of 0-4, the anti IL-12 treatedmice had a histology score of only 0.5 indicating a nearly completeresolution of psoriatic lesions FIG. 8. Therefore 2 injections ofanti-IL-12 were shown to be an effective treatment by clinicalobservations and by histology, shown in FIG. 5. Treatment began 9 weeksafter T cell transfer, injections given on week 1 and 2, all animalsreceived 1 mg/dose. Control animals were left untreated, isotype controlanimals received an isotype matched monoclonal antibody against a humanantigen. p=0.02.

[0105] An experimental antibody against a mouse antigen (BSK001) wastested, and found to have no effect on psoriasis lesions. The skinthickness on week 5 of disease did not decrease, as shown in FIG. 6.Each group consisted of 5 mice and all were induced with psoriasis bystandard protocol. The treatment for each group was 1 mg of antibody perdose: positive control (anti IL-12 mAb), negative control (isotypematched anti-human antigen mAb), and the experimental (mAb labeled BSKAb001), began on the third week, after disease had begun with moderateto severe psoriasis. Where as the mice in the positive control groupdemonstrated reduced skin thickness (7 μm improvement), the experimentalmAb BSK Ab001 group of mice had an increase in ear thickness of 19 μm 2weeks after the final injection, p=5.0×10⁻⁹. The isotype negativecontrol mice completed the treatment and observation period (total of 4weeks) with no change in ear thickness.

[0106] These results were confirmed by histology scores composed of theaverage of histology scores given to each ear of the experimental groupshistology. Semiquantitive histological scores from 0 to 4 were givenbased on the severity of inflammation. Initial histological evaluationwas performed by an independent outside pathologist. In later studiesevaluation was blindly conducted by three different investigators. 0=nosigns of inflammation; 1=very low focal areas of infiltration, mildacanthosis; 2=low level of mononuclear cell infiltration, mildthickening of epidermis, mild to moderate acanthosis 3=high level ofmononuclear cell infiltration, high vascular density, thickening of theepidermis (acanthosis, rete pegs and hyperplasia of epidermis andkeratinocytes, microabscesses, thinning of the granular cell layer4=very extensive infiltration in epidermis and dermis, very highvascular density, extreme thickening of epidermis, pustule formation anddestruction of granular cell layers.

[0107] In another study, it was found that treatment with anti CD43 mAbshowed no effect on the progression of disease (see FIG. 7). In thisexperiment all mice were induced with disease by standard protocol. Fivemice per group were utilized to compare the affects of anti-CD43 tountreated mice. The treated group received a treatment regimen of 2injections on week 7 and 8, of 1 mg per mouse mAb given sc. The micewere observed for 2 weeks after the final injection and no significantdifferences between the groups were found as observed by ear thickness.Anti CD43 was selected as a possible therapeutic mAb due to previousstudies that showed this antibody has the ability to abrogatesemi-chronic diseases and to prevent the induction of disease. Due tothe lack of improvement in disease severity in our model we determinedthat anti CD43 mAb is not an effective treatment in truly chronicdisease models. From this we show that the model is selective and doesnot react to all antibodies against mouse antigens.

[0108] Animals were tested to compare the effect of an experimental oralcompound (BSK 002) on the induction and severity of psoriatic disease.Twenty animals were induced with psoriasis by the standard protocol andwere divided into 2 groups: autoclaved water treated with BSK 002, andnormal autoclaved water. The effect of compound BSK002 was compared to anegative control at week 4, and it was found that 33% (3/9) of animalswithout BSK 002 compared to 0% (0/10) mice with BSK002 showed a diseasestate (ear thickness >=25 μm). At week nine, while both groups haddiseased animals, the group without BSK002 administration developed ahigher severity (30 μm vs 24 μm) as well as higher penetrance 100% (9/9)compared to 50% (5/10). Hence the presence of the experimental compoundBSK002 slows down the onset of psoriasis and reduces both penetrance andseverity (shown in FIG. 8). On week 11 all animals had disease (100%;10/10) in the normal water group while the treated water group had 80%(8/10). At week 9: p=1.7×10⁻⁵, week 11: p=3.4×10⁻², week 17: p=4.3×10⁻².

[0109] Animals were tested to determine the effect of cyclosporin A onthe development of disease (shown in FIG. 9), with injections of thecompound at 17, 18 and 19 weeks post-T cell transfer. It was found thatthe presence of the cyclosporin A reduced the severity of the disease,p=3.6×10⁻².

[0110] The known immunosuppressant methylprednisone was also found tocontrol the disease. After injection with methylprednisone, at a dose of40 mg/kg twice/day for 8 days, the ear thickness improved immediatelycompared to the control animals which received injections of PBS. After8 days of treatment the skin thickness was reduced from 33.7 to 22.4 μmp=2.01×10⁻⁶

[0111] To demonstrate that the disease could be induced at a site otherthan skin, a study was conducted in which CD25 negative effector cellswere transferred into scid/scid mice without co-injection into the skin.Food and gut flora are acting as co-factor antigen(s) in this modelset-up. After, 6-8 weeks animals developed severe colitis as measured byweight (average weight 15.8±0.6 (n=4), Normal weight is 19-23).

What is claimed is:
 1. A non-human mammal comprising: exogenousimmunocompetent effector cells, wherein said effector cells weredepleted of cells expressing CD25 prior to introduction into saidnon-human mammal; antigen presenting cells to which said immunocompetenteffector cells are tolerant and which are capable of initiating aninflammatory response by said immunocompetent effector cells; andinflamed tissue as a result of said inflammatory response.
 2. Thenon-human mammal according to claim 1, wherein said immunocompetenteffector cells comprise human T cells.
 3. The non-human mammal of claim2, wherein said T cells comprise CD4⁺ T cells.
 4. The non-human mammalof claim 3, wherein said mammal is a rodent.
 5. The non-human mammal ofclaim 4, wherein said rodent is a mouse.
 6. A panel for compoundtesting, comprising at least mammals according to claim 1, wherein atleast one of said mammals comprises a known immunomodulatory compound,and at least one of said mammals comprises a test compound suspected ofimmunomodulatory activity.
 7. A method for inducing chronic inflammationin an non-human mammal, the method comprising: transferring a cellpopulation comprising immunocompetent effector cells and lacking CD25positive T cells, from a donor non-human mammal to an immunocompromisednon-human mammal host, wherein said immunocompetent effector cellpopulation is tolerant of the host major histocompatibility antigens butis immunoreactive with one or more antigens present in said host;wherein said host develops chronic inflammation.
 8. The method accordingto claim 7, wherein said immunocompetent effector cells comprise Tcells.
 9. The method according to claim 8, wherein said T cells compriseCD4+ T cells.
 10. The method according to claim 9, further comprisingadministering an immunostimulatory co-factor to said mammal.
 11. Themethod according to claim 9, wherein said CD4⁺ T cells are reactive tominor histocompatibility antigens present in said host.
 12. The methodaccording to claim 10, wherein said immunostimulant is administered at atargeted site, and said chronic inflammation develops at said targetedsite.
 13. The method of claim 7, wherein said host is a rodent.
 14. Themethod of claim 13, wherein said rodent is a scid-scid mouse.
 15. Themethod of claim 10, wherein said immunostimulant is a non-replicatingvirus.
 16. The method of claim 15, wherein said virus is an adenovirus.17. The method of claim 10, wherein said immunostimulant is animmunostimulatory oligonucleotide sequence.
 18. The method of claim 10,wherein said immunostimulant is a polyclonal activating agent.
 19. Themethod of claim 18, wherein said polyclonal activating agent is anendotoxin.
 20. T he method of claim 18, wherein said polyclonalactivating agent is a superantigen.
 21. The method of claim 20, whereinsaid superantigen is a bacterial superantigen.
 22. A method forscreening a candidate therapy for efficacy in treatment of chronicinflammation, the method comprising: transferring a cell populationcomprising immunocompetent effector cells and lacking CD25 positive Tcells, from a donor non-human mammal to an immunocompromised non-humanmammal host, wherein said immunocompetent effector cell population istolerant of the host major histocompatibility antigens but isimmunoreactive with one or more antigens present in said host; whereinsaid host develops chronic inflammation; treating said animals with saidcandidate therapy; determining the severity of disease in the presenceof said therapy, wherein a decrease in severity of disease in thetreated animals relative to control animals is indicative of efficacy intreatment.
 23. The method according to claim 22, wherein saidimmunocompetent effector cells comprise T cells.
 24. The methodaccording to claim 23, wherein said T cells comprise CD4+ T cells. 25.The method according to claim 22, further comprising administering animmunostimulatory co-factor to said mammal.
 26. The method according toclaim 22, wherein said CD4⁺ T cells are reactive to minorhistocompatibility antigens present in said host.
 27. The methodaccording to claim 10, wherein said immunostimulant is administered at atargeted site, and said chronic inflammation develops at said targetedsite.
 28. The method according to claim 22, wherein said candidatetherapy comprises administration of one or a combination of candidateimmunosuppressant drugs.
 29. The method according to claim 22, furthercomprises comparison of said disease severity to a positive controlanimal treated with a known immunomodulatory compound.